Preparation and evaluation of [ 125 I]troxacitabine: L-nucleoside model of a potential agent for tumor diagnosis and radiotherapy O. A. El-Kawy, aà A. M. Hashem, b M. A. Amin, b and A. S. El-Wetery a In this study, the optimization of troxacitabine labeling with iodine-125 and its biological evaluation were described. Troxacitabine was labeled via direct electrophilic substitution using chloramine-T as oxidizing agent. The optimum amounts of reactants were: 50 lg troxacitabine, 75 lg Chloramine-T and 19 kBq carrier free Na 125 I. The labeled troxacitabine was stable for more than 24 h. Results of the in-vivo evaluation revealed that the new tracer, [ 125 I]troxacitabine, tends to localize in tissues with high proliferation rate with preferential accumulation in cancerous tissues. Imaging should be carried at 3 h postinjection. The in vitro cell growth inhibition assay showed that the effect of [ 125 I]troxacitabine was stronger than the effect of cold troxacitabine, which strongly suggested that its cytotoxicity was mainly due to radiotoxicity rather than chemotherapeutic activity. The binding assay revealed that [ 125 I]troxacitabine uptake by the Ehrlich and the ARAC8C cells was high and that it bounded well to DNA. Keywords: troxacitabine; auger-electron emitters; iodine-125; tumor targeting; radionuclides therapy Introduction 125 I-labeled nucleoside analogs, such as deoxyuridine, 1 cytar- abine 2 and gemcitabine 3 , have attracted the attention as tumor imaging and therapeutic agents. The iodinated compounds behave like thymidine due to the similarity of Van der Waals radii. 4 Once within the cell, the compounds undergo chemical phosphorylation and are incorporated in DNA of dividing cells. The decay of those auger electron emitters in close proximity to (or within) the DNA would deliver a radiation introducing highly toxic double breaks in their genomes. In addition to that, process of 125 I decay by electron capture leaves the resulting 125 Te with a mean net charge of18 interfering with the DNA repair process. The cytotoxicity of these labeled compounds can be attributed to the combination of their radiotoxic and chemotherapeutic activities. 3 However, their uptake is mediated through nucleoside transporter, thus they are expected to be of a little importance in malignancies that lack or have low nucleoside transport activity. 5 Naturally occurring nucleosides and nucleoside analogs developed to date as anticancer therapeutics are in the D stereochemical configuration. Until recently, corresponding unnatural L-enantiomers have largely been considered to be unrecognizable by cellular enzymes and therefore biologically inactive. 6 Troxacitabine [()-2V-deoxy-3V-oxacytidine; Troxatyl TM ] is a novel unnatural L-nucleoside analog with in-vitro and in-vivo antitumor activity against human leukemias and a variety of solid tumors, including hepatocellular, ovarian, prostate and renal carcinomas. 7–13 The different isomeric configuration confers to troxacitabine different mechanistic properties relative to D-nucleosides: its major route of cellular uptake is noncarrier mediated passive diffusion and deficiencies in nucleoside transport are unlikely to impart resistance to it, it is resistant to catabolic deamination and is highly effective against MDR tumors exhibiting high levels of cytidine deaminase, which represents an inactivation step for other nucleoside ana- logs. 5,8,14,15 Troxacitabine is phosphorylated from diphosphate to triphosphate by 3-phosphoglycerate kinase, and excised by apurinic/apyrimidinic endonuclease (APE1), an enzyme respon- sible for DNA repair and regulation of gene expression. 5,8,14,15 It causes complete DNA chain termination as it lacks a hydroxyl group and is a potent inhibitor of DNA polymerases. 5,16 In this study, troxacitabine was labeled with 125 I and the parameters affecting this labeling reaction were investigated to select the optimum conditions required to produce high labeling yield with high purity. Biological evaluation of the labeled troxacitabine was carried in-vivo in normal and tumorized mice. In vitro cell growth tests and a DNA binding assay were carried out. 98 a Atomic Energy Authority, Cairo, Egypt b Faculty of Pharmacy, Cairo University, Giza, Egypt *Correspondence to: O. A. El-Kawy, 11 Lateef street, Giza 12551, Egypt. E-mail: elkawyo@gmail.com Research Article Received 6 October 2009, Revised 22 June 2010, Accepted 24 June 2010 Published online 16 September 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jlcr.1820 J. Label Compd. Radiopharm 2011, 54 98–104 Copyright r 2010 John Wiley & Sons, Ltd.